Name: using rna-mediated interference feeding strategy to screen for genes involved in body size regulation in the nematode c. elegans
Uploaded: 2013-02-13T12:00:00
Description: Watch this Scientific Journal Video about Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans at JoVE.com

我们感谢詹姆斯·C.克拉克为动物提供的数字，在不同的发育时间点。 的菌株在这项研究中获得的秀丽隐杆线虫的遗传学中心，这是由美国国立卫生研究院国家研究资源中心（NCRR）的支持。这项工作是从纽约市立大学支持的CIRG 1817 JL和惩教署和美国国立卫生研究院1R15GM073678-01和1R15GM097692-01，惩教署，我们感谢William水稻博士，博士娜塔莉亚·霍尔茨曼，梅丽莎Silvestrini的手稿上的评论。这项工作是进行在部分履行一个博士学位研究生中心纽约市立大学（S.雄）“的要求。

1。准备携带目的基因序列的RNA干扰进料板<ol><li>如果使用市售的RNAi库（ 例如，从源生物科学生命科学），继续执行步骤1.4。另外，克隆到载体L4440，一种常用的蠕虫RNAi质粒8的靶基因序列，通过标准的克隆协议。 </li><li>变换的菌株HT115（DE3）中，用25μg/ ml的羧苄青霉素的LB琼脂平板上培养和12.5微克/毫升四环素的重组质粒转化，并挑选单个克隆，以备将来使用。 </li><li>同时，转换...

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在这个协议中，我们描述的车身尺寸有缺陷的突变体C.识别方法通过RNA干扰喂养的线虫 。此方法是适用于TGF-β信号分量的识别。由于这样的组件通过进化15是高度保守的，这样的屏幕是在所有后生动物阐明的分子机制的TGF-β信号有关。在这样的屏幕设计是一个重要的考虑出发菌株。我们已经证明，无论ERI-1，LIN-15b和RRF-3更敏感的RNAi喂养比对照菌株，这与以往的研...

<table border="1"><tbody><tr><td></td><td></td><td></td><td> - RNA干扰蠕虫板 17.7克WORM介质混合 60克琼脂 H 2 O 3升。高压灭菌。 加入3毫升1M IPTG（无菌）和3毫升25毫克/毫升羧苄青霉素（无菌）;然后倒入培养皿60毫米。 蠕虫板 17.7克WORM介质混合 0.6克硫酸链霉素 60克琼脂 H 2 O 3升。高压灭菌。倒成60毫米的培养皿中。 WORM介质混合 的55克Tris-CL 的24克Tris-OH 310克细菌蛋白胨 800毫克胆固醇 200克氯化钠 调匀，一定要避免大块，可以存放许多个月前使用这种粉状混合物。 2％琼脂糖 0.5 G琼脂糖 广告D 25毫升蒸馏水2 O。在微波加热，直至溶解。 1M异丙基β-D-1-硫代吡喃半乳糖苷（IPTG）诱导 2克IPTG 毫升蒸馏水溶解IN10 2 O 1M NaN的3 6.5克楠3 添加蒸馏水至100毫升2 O 25毫米楠3 2.5毫升1M南3 添加蒸馏水至100毫升2 O 秀丽隐杆线虫线虫遗传学中心 L4440质粒（携带氨苄青霉素抗性基因） 细菌菌株HT115（DE3）中（包含经IPTG诱导T7聚合酶; RNA酶III基因（dsRNAse）也携带四环素抗性基因的缺陷）16 60毫米陪替氏培养皿 100毫米培养皿 14毫升的园底猎鹰文化管 玻璃幻灯片 盖玻片 1-20微升移液器 20-200微升移液器 200-1000微升移液器 1-200μL枪头 200-1000微升枪头 1.5毫升的Eppendorf管 铂丝蠕虫挑</td></tr></tbody></table>

using rna-mediated interference feeding strategy to screen for genes involved in body size regulation in the nematode c. elegans

双链RNA介导的干扰（RNAi）是一种有效的策略，以击倒靶基因的表达1-3。它已被应用到许多模型系统，包括植物，无脊椎动物和脊椎动物。有各种各样的方法来实现RNAi 在体内 4,5。例如，可以与靶基因的RNAi载体转化，然后永久或瞬时转化的细胞系或初级细胞实现的基因敲除效果，或者从特定的靶基因（RNAi的寡核苷酸）的合成的双链寡核苷酸，可以是瞬时转化靶基因沉默的细胞系或原代细胞或合成的双链RNA分子可显微注射到生物体。由于线虫C.的利用细菌作为食物来源，饲养的动物与对靶基因的表达双链RNA的细菌提供了一个可行的策略。在这里，我们提出的RNAi输送机身尺寸表型的方法来得分。车身尺寸C.线虫主要受型TGF-β -等配位体DBL-1，所以该法是适合识别的TGF-β信号元件7。我们使用不同的菌株，其中包括两个RNA干扰过敏株重复供给的RNAi实验。我们的研究结果表明，RRF-3株给了我们最好的预期的RNAi表型。该方法易于操作，重现性好，易于量化。此外，我们的协议，最大限度地减少了使用专门的设备，所以它是适合于规模较小的实验室或主要是本科院校。

在我们的研究中，我们专注上机身尺寸调节的DBL-1/TGF-β途径。 DBL-1途径体型小，更短的车身长度相比，与野生型动物7,10,11功能的损失。因此，屏幕为C.线虫的机身尺寸突变体能够确定TGF-β信号组件和改性剂。 DBL-1信号是介导的一个保守的TGF-β的信号转导途径，其中包括细胞表面受体与细胞内的Smad信号传感器12。要测试的有效性的RNAi通过喂养的车身尺寸突变体的识别，我们?...

Watch this Scientific Journal Video about Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans at JoVE.com

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

我们演示了如何使用供给的RNAi技术敲除目标基因和分体尺寸型<em&gt; C.线虫</em&gt;。这种方法可用于大规模的画面，以找出潜在的基因成分，如在车身尺寸上的DBL-1/TGF-β信号调节。

使用屏幕在车身尺寸规例中的线虫基因介导的RNA干扰取食策略<em&gt; C.线虫</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Research

JoVE Journal

Biology

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

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科研

生物学

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